Cytotoxic  t lymphocyte

ABSTRACT

The present invention relates to a T lymphocyte having an activity to induce a T lymphocyte recognizing an antigen and a technique to use the T lymphocyte.

The present application is a Division of U.S. patent application Ser.No. 2/113,822, filed on May 1, 2008, which is a Division of U.S. patentapplication Ser. No. 11/171,365, filed on Jul. 1, 2005, now abandoned.U.S. patent application Ser. No. 11/171,365 (as well as the instantapplication) claims the benefit of priority of Japanese PatentApplication No. 2004-290785 filed on Oct. 1, 2004. The entire contentsof the above-identified applications are herein incorporated byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to T lymphocytes having an activity ofinducing T lymphocytes which recognize an antigen, a method of inducingspecific antigen-specific T lymphocytes, use of induced T lymphocytes asa therapeutic agent for cancer or an infectious disease,HLA-A2402-restricted cytotoxic T lymphocytes (CTLs) specific for atumor-associated antigen, an antigen peptide recognized by the CTL, useof the antigen peptide as a CTL inducer and a therapeutic agent forcancer, and a tetramer formed by tetramerizing MHC/antigen peptidecomplexes which is useful for detection of the CTL.

2. Discussion of Related Art

Among cytotoxic T lymphocytes (CTLs), there is a CTL capable ofrecognizing, by a specific T cell receptor (abbreviated hereinafter as“TCR”), a complex wherein an antigen peptide and a majorhisto-compatibility antigen MHC molecule encoded by a majorhisto-compatibility gene complex (abbreviated hereinafter as “MHC”) arebound to each other, thus injuring cells presenting the complex on thecell surface thereof. The major histo-compatibility antigen MHCmolecule, in the case of humans, is called human leukocyte antigen(abbreviated hereinafter as “HLA”). The CTL recognizes and injures onlya target cell having the same HLA molecule as in the CTL itself.Accordingly, the CTL is called “HLA-restricted CTL”.

The cytotoxicity reaction can be generated by:

1) the presence of a CTL having a specific TCR, and2) the presence of an antigen peptide not only capable of binding to anHLA molecule but also forming a complex recognized by the TCR in orderto become an antigen peptide presented by an HLA and recognized by theCTL.

Such antigen peptide is generated, for example, by processing, in anendoplasmic reticulum, of an antigen and the like such as a proteinsynthesized intracellularly in a mammalian cell, to degrade the antigenand the like into smaller epitope peptides. The antigen peptide isfurther associated with an HLA molecule and presented on the surface ofa cell. I.e., the protein is degraded into peptides consisting of from 8to 15 amino acid residues in a proteosome complex consisting of manysubunits, and some of the generated peptides are transported from thecytosol to an endoplasmic reticulum by a TAP transporter. The peptides,when bound to a class I/β2 microglobulin heterodimer in the endoplasmicreticulum, are stabilized as a 3-molecule complex and transportedthrough a Golgi apparatus into the surface of a cell.

Furthermore, it has been revealed that upon infection with organismssuch as viruses, microorganisms, protozoa and fungi, a CTL against anantigen possessed by these organisms plays an important role inprotection against infection.

The HLA class I molecules are roughly divided into HLA-A, HLA-B andHLA-C. It is known that an antigen peptide presented upon binding to theHLA class I molecule is composed of from 8 to 10 amino acid residues andhas certain structural features which vary depending on the respectiveHLA molecules. For example, a peptide consisting of from 9 to 10 aminoacid residues having a Leu residue at the second position from theN-terminal thereof and a Leu residue or a Val residue at the C-terminalis best known worldwide as a peptide binding to the HLA-A2.1 moleculefound most frequently. A peptide consisting of from 9 to 10 amino acidresidues having any one of a Tyr residue, a Phe residue, a Met residueand a Trp residue at the second position from the N-terminal thereof andany one of a Leu residue, an Ile residue, a Trp residue and a Pheresidue at the C-terminal is best known as a peptide binding to anHLA-A24 molecule abundant in Asians races including Japanese (J.Immunol., 155, p. 4307-4312 (1995)).

Tumor antigens of which antigen peptides have been identified up to nowinclude MAGE-1 and MAGE-3 against HLA-A1; MAGE-3, MART 1, tyrosinase,gp100, HER2/neu, CEA and the like against HLA-A2.1; MAGE-3 againstHLA-Cw1; MAGE-3 against HLA-B44; MAGE-A4 against HLA-B37; MAGE-1,MAGE-2, MAGE-3, CEA, HER2/neu, tyrosinase and β-catenin against HLA-A24,and the like.

Many of the antigen peptides have been found by establishing a tumorcell-recognizing class I-restricted CTL, to identify a tumor antigenrecognized by the CTL, finding the minimum unit in a protein serving asthe tumor antigen by a genetic engineering method and selecting apeptide in the minimum unit, on the basis of information on a bindingmotif to HLA class I molecule (Proc. Natl. Acad. Sci. USA, 91, p.3515-3519 (1994)).

The antigen peptide is determined by finding HLA class Imolecule-binding peptides consisting of a sequence in a tumor antigenprotein, on the basis of a motif structure common in the HLA class Imolecule-binding peptide, selecting a CTL-inducible peptide from thefound HLA class I molecule-binding peptides, by using antigen-presentingcells, and evaluating whether a CTL having cytotoxicity on tumor cellscan be finally induced or not (Proc. Natl. Acad. Sci. USA, 91, p.2105-2109 (1994); J. Exp. Med., 179, p. 921-930 (1994)).

The HLA class I molecules are classified into some subtypes. The subtypepossessed by humans varies significantly among races. The proportion ofhumans having HLA-A2 is highest in the world and accounts for 45% of theCaucasoid, for example. Identification of the HLA-A2-restricted antigenpeptide has advanced most. On the other hand, in the Japanese, theproportion possessing HLA-A2 accounts for 40%. 20% of the Japanese haveHLA-A*0201 which is the same subtype as in the Caucasoid, and many ofthe rest of the Japanese have A*0206. Peptides binding to these subtypesvary depending on the subtype. For example, a mainly studied peptidebinding to HLA-A2 is HLA-A*0201. On the other hand, the proportion ofthe Japanese possessing HLA-A24 accounts for 60% or more. The proportionpossessing the HLA-A24 is higher in Asian races than in other races.

The antigen peptide, even if the antigen is the same, varies dependingon the type of HLA, and induction of a CTL utilizing the antigen peptideis thus troublesome. To solve this problem, various devices have beenmade, but satisfactory results have not been obtained yet at present.One of the devices is a method of inducing T lymphocytes by utilizingcells obtained by transducing an antigen gene into antigen-presentingcells derived from a patient himself (autologous). As theantigen-presenting cells, use of B cells, macrophages or dendriticcells, known as professional antigen-presenting cells, have beenexamined, and a clinical test of using the dendritic cells mainly forvaccine adjuvant and the like is conducted (J. Immunotherapy, 21, p.41-47 (1998)). In these antigen-presenting cells, however, there aresome disadvantages that much labor is required to prepare the cells in anecessary amount to induce immunization. In addition, the B cells havethe advantage that the cells can be prepared in a large amount byimmortalization with EB virus. Owing to use of the virus, however, thereis the disadvantage of lacked generality. With respect to the method ofintroducing the gene, introduction of the gene using a virus vector or aplasmid DNA has the disadvantage of generating a new variant in somecases by insertion of the gene into a chromosome.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to provideRNA-introduced T lymphocytes which enable induction of cytotoxic Tlymphocytes which can recognize an antigen, for example act specificallyon organisms causing an infectious disease or tumor cells inindividuals, exhibit a therapeutic action on a tumor, causing specificcytolysis of target cells or cells presenting a specific antigen in theindividuals, or cause cytokine release reaction. A second aspect of thepresent invention relates to provide an immune inducer capable ofinducing the cytotoxic T lymphocytes or inducing immunity effectiveagainst cancer or an infectious disease in the individuals. A thirdaspect of the present invention relates to provide a method of inducingT lymphocytes, which at least enables induction of T lymphocytesrecognizing an antigen of interest. A fourth aspect of the presentinvention relates to provide a therapeutic agent for cancer or aninfectious disease, which acts specifically on organisms causing aninfectious disease or tumor cells in the individuals. A fifth aspect ofthe present invention relates to provide cytotoxic T lymphocytes whichenable recognition of an antigen, causing specific cytolysis of targetcells or cells presenting a specific antigen, or causing cytokinerelease reaction, in the individuals. A sixth aspect of the presentinvention relates to provide an inducer of the cytotoxic T lymphocytes,which enables any one of induction of the cytotoxic T lymphocytes orexhibiting a specific action on organisms causing an infectious diseaseor tumor cells in the individuals. A seventh aspect of the presentinvention relates to provide a tetramer for detecting T cell receptorspossessed by cytotoxic T lymphocytes, which enables any one ofmonitoring of the cytotoxic T lymphocytes in the individuals or samplesderived from the individuals.

A first embodiment of the present invention relates to RNA-introduced Tlymphocytes into which an RNA encoding an antigen of interest isintroduced, the RNA-introduced T lymphocytes having an activity ofinducing T lymphocytes which recognize the antigen. Such RNA-introducedT lymphocytes include, for example, CD4-positive cells activated byphytohemagglutinin. Such RNA-introduced T lymphocytes have an excellenteffect that, for example, cytotoxic T lymphocytes which recognize anantigen can be induced by a simple technique. Here, such cytotoxic Tlymphocytes can act specifically on organisms causing an infectiousdisease or tumor cells in individuals, can exhibit a therapeutic actionon a tumor, and can specifically cause cytolysis of target cells orcells presenting a specific antigen, cytokine release reaction and thelike, in the individuals.

A second embodiment of the present invention relates to a method ofinducing T lymphocytes which recognize the antigen, which comprisesusing the RNA-introduced T lymphocytes in the first embodiment asantigen-presenting cells to induce T lymphocytes which recognize theantigen. The RNA-introduced T lymphocytes include, for example,CD4-positive cells activated with phytohemagglutinin. In addition, the Tlymphocytes which recognize an antigen include, for example,CD8-positive cytotoxic T lymphocytes. The antigen includes atumor-associated antigen or an antigen of an infectious microorganism.The RNA includes, for example, an RNA prepared from cancerous tissues.The antigen includes, for example, EBNA3A, CMVpp65 and the like. Thisinduction method exhibits an excellent effect that T lymphocytesrecognizing the antigen of interest can be simply induced.

A third embodiment of the present invention relates to an immuneinducer, which comprises the RNA-introduced T lymphocytes in the firstembodiment. Such immune inducer exhibits an excellent effect that, forexample, the cytotoxic T lymphocytes can be induced to induce immunityeffective against cancer or an infectious disease in the individuals.

A fourth embodiment of the present invention relates to a therapeuticagent for cancer or an infectious disease, which comprises, as an activeingredient, T lymphocytes obtained by the method of inducing Tlymphocytes in the second embodiment. Such therapeutic agent for canceror an infectious disease exhibits an excellent effect that the agent canact specifically on, for example, organisms causing an infectiousdisease or tumor cells in the individuals.

A fifth embodiment of the present invention relates to cytotoxic Tlymphocytes which recognize cells presenting, on the surfaces of thecells, a complex of a human major histo-compatibility antigen(HLA)-A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2and an HLA-A24 molecule, or a complex of a functional derivative of theHLA-A24-restricted antigen peptide and an HLA-A24 molecule and which arepositive to CD8. Such cytotoxic T lymphocytes exhibit an excellenteffect of specific recognition of an antigen. The cytotoxic Tlymphocytes of the present invention also exhibit an excellent effectthat the cytotoxic T lymphocytes can act specifically on, for example,tumor cells in the individuals, exhibit a therapeutic action on tumor,and cause specific cytolysis of target cells or cells presenting aspecific antigen, cytokine release reaction and the like, in theindividuals.

A sixth embodiment of the present invention relates to a therapeuticagent for cancer, which comprises the cytotoxic T lymphocytes in thefifth embodiment as an active ingredient. Such therapeutic agent forcancer exhibits an excellent effect that, for example, it can actspecifically on tumor cells in the individuals.

A seventh embodiment of the present invention relates to an inducer forthe cytotoxic T lymphocytes in the fifth embodiment, which comprises, asan active ingredient, at least one antigen peptide selected from thegroup consisting of a human major histo-compatibility antigen(HLA)-A24-restricted antigen peptide represented by SEQ ID NO: 1 or 2and the functional derivative thereof. Such inducer exhibits anexcellent effect that, for example, the cytotoxic T lymphocytes can beinduced by a simple technique to exhibit a specific action on tumorcells in the individuals.

An eighth embodiment of the present invention relates to a therapeuticagent for cancer, which comprises, as an active ingredient, at least oneantigen peptide selected from the group consisting of a human majorhisto-compatibility antigen (HLA)-A24-restricted antigen peptiderepresented by SEQ ID NO: 1 or 2 and the functional derivative thereof.Such therapeutic agent for cancer exhibits an excellent effect that, forexample, the agent can act specifically on tumor cells in theindividuals.

A ninth embodiment of the present invention relates to a tetramer fordetecting T cell receptors possessed by the cytotoxic T lymphocytes inthe fifth embodiment, which comprises a human major histo-compatibilityantigen (HLA)-A24-restricted antigen peptide represented by SEQ ID NO: 1or 2 or the functional derivative thereof. Such tetramer exhibits anexcellent effect that, for example, the tetramer can monitor thecytotoxic T lymphocytes in the individuals.

According to the present invention, there is provided a method ofinducing a CTL, wherein easily obtainable T lymphocytes are used asantigen-presenting cells. Further, there is provided a novelHLA-A24-restricted antigen peptide. The CTL induced by using theantigen-presenting cells or the antigen peptide is useful, for example,in treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C are graphs showing immunogenicity of peptides inHHDA2402+/−β2m−/− mice. FIG. 1A shows the results of EBNA3A, FIG. 1Bshows the results of MAGE-A4, and FIG. 1C shows the results of SAGE.

FIG. 2 is a graph showing immunogenicity of MAGE-A4-derived peptides inhumans. Panel A is a graph showing the results of IFN-γ ELISPOT assay ofhuman bulk CTLs sensitized twice with autologous CD4+ PHA blast cellsinto which a mRNA corresponding to MAGE-A4 or SAGE was introduced. Inpanel A, the black bar indicates LCL into which MAGE-A4 mRNA wasintroduced, and the shaded bar indicates LCL into which SAGE mRNA wasintroduced. Panel B is a graph showing the results of expansion of well#2 with autologous LCL into which mRNA corresponding to MAGE-A4 or SAGEwas introduced, or with autologous PBMC and IL-2 (20 IU/milliliter).

FIG. 3 is a graph showing the results of presentation of cell surfaceswith MAGE-A4₁₄₃₋₁₅₁ peptide together with HLA-A2402 after intracellularprocessing. Panel A shows the result of staining with MAGE-A4₁₄₃₋₁₅₁ A24tetramer, and panel B shows the results of staining with a controltetramer. Panel C shows the cytotoxicity of MAGE-A4₁₄₃₋₁₅₁-specific CTL#2-28 cells on tumor cells. The black triangle shows T2A24 pulsed withMAGE-A4₁₄₃₋₁₅₁; the black square shows T2 pulsed with MAGE-A4₁₄₃₋₁₅₁;the lozenge shows T2A24 pulsed with SAGE₇₁₅₋₇₂₃; and the black circleshows T2A24 not pulsed. Panel D shows the cytotoxicity ofMAGE-A4₁₄₃₋₁₅₁-specific CTL #2-28 cells on tumor cells. The blacktriangle shows KE-4 cells expressing HLA-A2402 and MAGE-A4; the blacksquare shows TE-10 cells expressing HLA-A2402 and MAGE-A4; the lozengeshows 11-18 cells expressing HLA-A2402 and MAGE-A4; and the black circleshows LB-23 cells expressing HLA-A2402, but not expressing MAGE-A4.

FIGS. 4A to 4C are graphs showing the immunogenicity of SAGE₇₁₅₋₇₂₃peptide in humans. FIG. 4A is a graph showing the results of IFN-γELISPOT assay using human bulk CTL cells obtained by sensitization twicewith autologous CD4+ PHA blast cells into which SAGE mRNA wasintroduced. In FIG. 4A, the black bar indicates the results where T2A24cells pulsed with SAGE₇₁₅₋₇₂₃ peptide were used as target cells, and theshaded bar indicates the results where T2A24 cells pulsed with a controlpeptide (HER2₆₃₋₇₁) were used. FIG. 4B is a graph showing the results ofexpansion of bulk CTLs in well #2. FIG. 4C is a graph showing theresults of IFN-γ ELISPOT assay using human bulk CTL cells obtained bysensitization twice with autologous CD4+ PHA blast cells into which SAGEmRNA was introduced.

FIGS. 5A to 5C are graphs showing the results of presentation of SSAGE₇₁₅₋₇₂₃ peptide on cell surfaces together with HLA-A2402 afterintracellular processing. FIG. 5A is a graph showing the results ofstaining with SAGE₇₁₅₋₇₂₃ A24 tetramer. FIG. 5B is a graph showing theamount of IFN-γ released by SAGE₇₁₅₋₇₂₃-specific CTL #22 cells in thepresence of various cells. FIG. 5C shows the cytotoxicity ofSAGE₇₁₅₋₇₂₃-specific CTL #22 cells on various cells. In FIG. 5C, panel Ashows K562 cells not expressing SAGE or HLA-A2402. In panel A, thelozenge shows K562 cells, the black square shows K562A24 cells, and theblack triangle shows K564A24 pulsed with SAGE₇₁₅₋₇₂₃. Panel B shows R27cells expressing SAGE but not expressing HLA-A2402. In panel B, thelozenge shows R27 cells, the black square shows R27A24 cells, and theblack triangle shows R27A24 pulsed with SAGE₇₁₅₋₇₂₃. Panel C shows LCLnot expressing SAGE but expressing HLA-A2402. In panel C, the blacktriangle shows LCL into which SAGE mRNA was introduced, and the lozengeshows LCL into which EBNA3A mRNA was introduced.

FIGS. 6A to 6D are graphs each showing one example of the results ofdetection of SAGE₇₁₅₋₇₂₃-specific precursor in healthy normal humanPBMC.

DETAILED DESCRIPTION OF THE INVENTION

In the present specification, the amino acid residue is sometimesexpressed in three-letter or one-letter designation of an amino acid inaccordance with conventional nomenclature in biochemistry.

The first embodiment of the present invention relates to RNA-introducedT lymphocytes into which an RNA encoding an antigen of interest isintroduced and having an activity of inducing T lymphocytes recognizingthe antigen. The RNA-introduced T lymphocytes of the present inventionhave RNA encoding an antigen of interest introduced into them, and thusexhibit an excellent effect that T lymphocytes recognizing the antigencan be obtained. Accordingly, the RNA-introduced T lymphocytes exhibitan excellent effect that cytotoxic T lymphocytes acting specifically onorganisms causing an infectious disease or tumor cells in individuals,exhibiting a therapeutic action on tumors, and causing specificcytolysis of target cells or cells presenting a specific antigen, andcytokine release reaction, in the individuals can be induced by a simplemethod.

The RNA-introduced T lymphocytes of the present invention can beprepared for example by introducing an RNA encoding an antigen ofinterest into T lymphocytes derived from natural origin.

The “T lymphocytes derived from natural origin” includes, for example,CD3-positive cells derived from individuals. The “T lymphocytes derivedfrom natural origin” can be identified by using, for example, amonoclonal antibody against CD3 and, if necessary, a monoclonal antibodyagainst a T cell antigen receptor. Such CD3-positive cells may also beCD4-positive and/or CD8-positive cells. The CD3-positive cells arepreferably CD4-positive.

The “antigen” includes, but not limited particularly to,tumor-associated antigens, antigens of infectious microorganisms, andthe like.

The “tumor-associated antigens” include MAGE (melanoma-associatedantigen) family protein, SAGE (sarcoma antigen), LAGE (L antigen),NY-ESO-1, WT-1, hTERT, and the like.

The “antigens of infectious microorganisms” include antigens of EB virussuch as EBNA-3A, antigens of cytomegalovirus such as CMVpp65, herpesvirus antigen, influenza virus antigen, HIV antigen, Salmonella antigen,Shigella antigen, Enterobacter antigen, protozoa-derived antigen,fungus-derived antigen, and the like.

The RNA may be an RNA prepared by usual genetic engineering techniques,or an RNA prepared from cells. The RNA can be obtained, for example, byextracting an RNA encoding an antigen of interest from cells,reverse-transcribing the resulting RNA into cDNA, amplifying theresulting cDNA by PCR, and carrying out transcription using theamplified DNA as a template. Such RNA can also be used for preparationof the RNA-introduced T lymphocytes of the present invention.

The method of introducing the RNA into T lymphocytes includes, forexample, physical methods such as electroporation, a particle gunmethod, a calcium phosphate method, a lipofection method and a liposomemethod; biological techniques using viral vectors such as a retrovirusvector, a lentivirus vector and an adenovirus vector; and the like.

Specifically, the RNA-introduced T lymphocytes of the present inventioninclude, for example, CD4-positive cells activated withphytohemagglutinin.

The second embodiment of the present invention relates to a method ofinducing T lymphocytes which recognize an antigen, which comprisesusing, as antigen-presenting cells (also referred to hereinafter as“APC”), RNA-introduced T lymphocytes into which an RNA encoding theantigen of interest is introduced, to induce T lymphocytes recognizingthe antigen. Such induction method is based on findings by the presentinventors that an antigen-specific CTL can be induced when autologouslymphocytes are stimulated with a T-cell activator such asphytohemagglutinin and cells into which an RNA encoding an antigen isintroduced by electroporation are used as antigen-presenting cells, andon surprising findings by the present inventors that the RNA-introducedT lymphocytes exhibit an ability to present the antigen.

The induction method of the present invention exhibits an excellenteffect that since the RNA-introduced T lymphocytes into which an RNAencoding the antigen of interest is introduced are used asantigen-presenting cells, T lymphocytes recognizing the antigen ofinterest can be simply induced. The induction method of the presentinvention is also advantageous in that the method is excellent inhandling (for example, collection, enlargement etc.) of cells ascompared with induction of a CTL with B cells, macrophages or dendriticcells known as professional antigen-presenting cells.

The “RNA encoding an antigen of interest” is exemplified by the similarRNA as illustrated in the first embodiment. Specifically, the RNAincludes, for example, an RNA encoding an antigen for which induction ofcytotoxic T lymphocytes which specifically recognize the antigen isdesired (antigen of interest). The antigen includes, for example, atumor-associated antigen or an antigen of an infectious microorganismand the like. The antigen of infectious microorganisms includes EBNA3A,CMVpp65 and the like. The RNA includes, for example, an RNA preparedfrom cancer tissues, and the like.

The method of artificially introducing an RNA encoding an antigen ofinterest into the RNA-introduced T lymphocytes used asantigen-presenting cells includes, but is not limited particularly to,the physical methods and biological methods described above. By suchartificial introduction methods, the RNA can be introduced into Tlymphocytes, to express the RNA in the resulting RNA-harboring Tlymphocytes.

The T lymphocytes and RNA-introduced T lymphocytes can be maintained,for example, in a medium such as RPMI, AIM-V and X-VIVO10, physiologicsaline, and buffer solutions such as phosphate buffered physiologicsaline.

In the induction method of the present invention, the RNA-introduced Tlymphocytes used as antigen-presenting cells may be CD3-positive cellsof an individual itself, for example, a patient himself/herself(autologous T lymphocytes) or CD3-positive cells derived from a donorhaving the same type of HLA as that of an individual such as a patient.The T lymphocytes, similarly to those in the first embodiment, may beCD4-positive or may be CD8-positive. The RNA-introduced T lymphocytesare preferably CD4-positive T lymphocytes. Here, when the individual isan individual, such as a patient, who has underwent transplantation suchas allogeneic hematopoietic stem cell transplantation, the T lymphocytesused can be T lymphocytes of a donor of the transplant.

The RNA-introduced T lymphocytes may be activated preferably with lectinsuch as phytohemagglutinin (PHA) and concanavalin (ConA) or an anti-CD3antibody in a medium containing IL-2, IL-7 and the like. Insofar as theactivation conditions are usually used conditions, the conditions arenot limited particularly. For use as antigen-presenting cells, theRNA-introduced T lymphocytes are treated by gamma ray irradiation orwith mitomycin.

Here, in the induction method of the present invention, theRNA-introduced T lymphocytes are preferably CD4-positive cells activatedwith phytohemagglutinin.

Specifically, the RNA-introduced T lymphocytes in the first embodimentcan be used as antigen-presenting cells to induce antigen-recognizing Tlymphocytes. The antigen-recognizing T lymphocytes to be induced includeCD8-positive cytotoxic T lymphocytes (CTLs) and CD4-positive helper Tlymphocytes. The “antigen-recognizing T lymphocytes to be induced” varydepending on a starting material used as a source of T lymphocytesderived from natural origin. The “starting material” includes, forexample, peripheral blood mononuclear cells (hereinafter also referredto as “PBMC”) collected from blood, and CD8-positive lymphocytesseparated from the PBMC by a method using an anti-CD8 antibody andmagnetic beads. For example, when the PBMC is used as the “startingmaterial”, lymphocytes usually in the form of a mixture ofantigen-recognizing CTLs and helper T lymphocytes are obtained. Inaddition, for example, when CD8-positive cells are used as the “startingmaterial”, lymphocytes containing a CTL are obtained.

When the CTL is to be induced in vitro, for example, the RNA-introducedT lymphocytes and a sample excised extracorporeally from a living bodyhaving the same type of HLA as in the RNA-introduced T lymphocytes canbe used to induce the CTL. The “sample excised extracorporeally” in thisspecification means a sample such as blood; lymph nodes, spleen andother various organs excised by an operation, and the like. Particularlyin the induction method of the present invention, lymphocytes andinfiltrating lymphocytes occurring in these samples are preferably used.

For example, when blood is used as the sample, the CTL of interest canbe induced by repetitive antigenic stimulation with the T lymphocytesinto which an RNA encoding the antigen of interest is introduced, tolymphocytes obtained from PBMC prepared from human blood having thecorresponding type of HLA. By cloning, the induced CTL can also bemaintained as lymphocytes having stabilized cytotoxicity. For example,the induced CTL can be proliferated by stimulation with feeder cells,antigen, various cytokines, or anti-CD3 antibody.

The activity of the induced “cytotoxic T lymphocytes which recognize anantigen”, i.e., the activity of the antigen-specific CTL, can beevaluated in terms of cytotoxicity on target cells as an indicator bymeasuring a radioactive substance released from the target cells pulsedwith a peptide from the CTL inducing antigen and labeled with aradioactive substance and the like. In addition, the activity of theantigen-specific CTL can be evaluated by measuring the incorporation ofradioactivity into the CTL in the presence of the antigen-presentingcells pulsed with the antigen peptide, whereby the proliferationreaction of the CTL against the antigen-presenting cells pulsed with theantigen peptide can be determined as an indicator of the activity of theantigen-specific CTL. Each of the target cells or antigen-presentingcells which are transduced with a DNA encoding the antigen or into whichan RNA encoding the antigen is introduced can also be used for the abovepurpose. The antigen-specific CTL can be detected by measuring theamount of cytokines such as GM-CSF and IFN-γ released in anantigen-specific manner from the CTL or the target cells. Theantigen-specific CTL can also be confirmed directly by using an antigenpeptide/HLA complex labeled with a fluorescent dye and the like. In thiscase, for example, the CTL can be contacted with a first fluorescentmarker coupled with a CTL-specific antibody, and the resulting productcan be contacted with an antigen peptide/MHC complex coupled with asecond fluorescent marker, to confirm the antigen-specific CTL bydetecting the presence of cells labeled with both the first and secondfluorescent markers by FACS (fluorescence-activated cell sorting)analysis.

The third embodiment of the present invention relates to an immuneinducer comprising the RNA-introduced T lymphocytes in the firstembodiment. The immune inducer of the present invention comprises theRNA-introduced T lymphocytes as an active ingredient, and thus exhibitsan excellent effect that the “cytotoxic T lymphocytes which recognize anantigen” capable of acting specifically on organisms causing aninfectious disease or tumor cells in individuals, exhibiting atherapeutic action on a tumor, and causing specific cytolysis of targetcells or cells presenting a specific antigen, and cytokine releasereaction, in the individuals, can be induced. The immune inducer of thepresent invention can induce immunity effective against cancer or aninfectious disease in the individuals.

The immune inducer of the present invention is provided in the form of asuspension of the RNA-introduced T lymphocytes in the first embodimentin a pharmaceutically acceptable diluent. Here, the “diluent” means, forexample, a medium suitable for storing the RNA-introduced T lymphocytesin the first embodiment, or physiologic saline or phosphate bufferedphysiologic saline. The medium generally includes, but not limitedparticularly to, a medium such as RPMI, AIM-V and X-VIVO10. Thesemediums are easily commercially available.

For the purpose of stabilization and the like, a pharmaceuticallyacceptable carrier may also be added to the immune inducer of thepresent invention. Here, in this specification, the carrier includes,for example, human serum albumin and the like.

The content of the T lymphocytes in the first embodiment in the immuneinducer of the present invention is desirably 1×10⁴ cells/milliliter ormore, preferably 5×10⁵ cells/milliliter or more, and 1×10⁸cells/milliliter or less, preferably 5×10⁷ cells/milliliter or less, pertype of T lymphocyte.

The immune inducer of the present invention is applied to bothadministration into a donor of the T lymphocytes (“autologousadministration”) and administration into another individual having thesame type of HLA (“allogenic administration”).

When the immune inducer of the present invention is to be administeredinto humans, the immune inducer of the present invention can beadministered subcutaneously, intracutaneously or intravenously by asyringe. Although the amount of the immune inducer of the presentinvention can be determined suitably depending on body weight, diseasestate, and the like, it is desired that the number of the RNA-introducedT lymphocytes administered into an adult is usually from 10⁶ to 10¹⁰cells per type of RNA-introduced T lymphocyte. The above range is astandard and not restrictive. Administration of the immune inducer ofthe present invention can be repeated until the desired effect isobtained.

The RNA-introduced T lymphocytes contained in the immune inducer of thepresent invention are T lymphocytes derived from an individual(specifically human) as the subject of administration or T lymphocyteshaving the same type of HLA as that of an individual as the subject ofadministration. Therefore, toxicity of the RNA-introduced T lymphocytesis not particularly recognized.

The immune inducer of the present invention exhibits an excellent effectthat a CTL recognizing an antigen encoded by an RNA introduced into theRNA-introduced T lymphocytes contained in the immune inducer is inducedin an individuals (specifically human) into which the immune inducer isadministered.

Accordingly, the pharmacological evaluation of the immune inducer of thepresent invention can be carried out, for example, by measuring theactivity of the CTL induced by the immune inducer of the presentinvention in terms of cytotoxicity on the target cells, proliferationreaction in the presence of antigen-presenting cells, the amount ofantigen-specific cytokines released, and the like.

The T lymphocytes obtained by the induction method in the secondembodiment, for example, the CTL, can be used as a therapeutic agent forcancer or an infectious disease. Accordingly, a fourth embodiment of thepresent invention relates to a therapeutic agent for cancer or aninfectious disease, which comprises, as an active ingredient, the Tlymphocytes obtained by the induction method in the second embodiment.The therapeutic agent can be formulated pursuant to a therapeutic agentfor cancer in a sixth embodiment described below, to use in treatment.

In this specification, the therapeutic agent for cancer is intended toencompass so-called carcinostatics.

Pharmacological evaluation of the therapeutic agent for cancer or aninfectious disease of the present invention, for example, in the case ofcancer, can be carried out where inhibition of growth of cancer cells,death of cancer cells, induction of cell death of cancer cells orshrinkage of cancer cells observed in examination of the cytotoxicity ofthe therapeutic agent on cancer cells, or shrinkage or disappearance ofa cancer site, or prevention of expansion of the cancer site, uponadministration of the therapeutic agent for cancer or an infectiousdisease into the cancer site or therearound, is used as an indicator ofthe effect of the therapeutic agent on cancer.

Pharmacological evaluation of the therapeutic agent for cancer or aninfectious disease of the present invention, for example, in the case ofan infectious disease, can be carried out by using, as an indicator ofthe effect of the therapeutic agent on an infectious disease,suppression of growth of an organism causing an infectious disease orits cells or death of the organism or its cells in examination of thecytotoxicity of the therapeutic agent on the organism or its cells, orreduction or disappearance of symptoms of an infectious disease, uponadministration of the therapeutic agent for cancer or an infectiousdisease in the present invention into individuals (e.g. humans) with aninfectious disease.

A fifth embodiment of the present invention relates to cytotoxic Tlymphocytes which recognize cells presenting a complex of a human majorhisto-compatibility antigen (HLA)-A24-restricted antigen peptiderepresented by SEQ ID NO: 1 or 2 and an HLA-A24 molecule, or a complexof a functional derivative of the antigen peptide and an HLA-A24molecule on the surfaces of the cells and which are positive to CD8.

The peptide of an amino acid sequence represented by SEQ ID NO: 1 is anHLA-A24-restricted antigen peptide derived from MAGE-A4. The peptide ofan amino acid sequence represented by SEQ ID NO: 2 is anHLA-A24-restricted antigen peptide derived from SAGE. Accordingly, theCTL of the present invention exhibits an excellent property ofspecifically causing cytolysis or cytokine release reaction of thetarget cells or antigen-presenting cells.

In this specification, the “functional derivative of theHLA-A24-restricted antigen peptide” means a substance having an abilityto form a complex with an HLA-A24 molecule, and the formed complex isrecognized by a CTL recognizing a complex of an antigen peptiderepresented by SEQ ID NO: 1 or 2 and an HLA-A24 molecule. The“functional derivative of the HLA-A24-restricted antigen peptide” is,for example, a peptide having an ability to form a complex with anHLA-A24 molecule, the formed complex being recognized by the CTL of thepresent invention, wherein the amino acid sequence of the functionalderivative is different from the amino acid sequence represented by SEQID NO: 1 or 2 by:

1) deletion,2) substitution with other amino acid residues or amino acid analogues,3) addition of one or more amino acid residues or amino acid analogues,or4) a combination thereofof one or several amino acid residues. The length of the amino acidsequence of the functional derivative is preferably from 9 to 10residues, but is not limited thereto particularly. The “amino acidanalogues” in this specification mean N-acylated amino acids, O-acylatedamino acids, esterified amino acids, amide amino acids, alkylated aminoacids, and the like.

Insofar as the complex of the HLA-A24-restricted antigen peptide or thefunctional derivative thereof and the HLA-A24 molecule is recognized bythe CTL of the present invention, a formyl group, an acetyl group, at-butoxycarbonyl (t-Boc) group or the like may be bound to theN-terminal amino group of the antigen peptide or the functionalderivative thereof, or to a free amino group of a side chain of an aminoacid residue. In addition, insofar as the complex of theHLA-A24-restricted antigen peptide or the functional derivative thereofand the HLA-A24 molecule is recognized by the CTL, a methyl group, anethyl group, a t-butyl group, a benzyl group or the like may be bound tothe C-terminal of the antigen peptide or the functional derivativethereof, or to a free carboxyl group in a side chain of an amino acidresidue thereof.

The functional derivative can be identified by using the CTL recognizinga complex of the antigen peptide represented by SEQ ID NO: 1 or 2 and anHLA-A24 molecule. The method of identifying the functional derivativeincludes, for example, the following methods.

The first method is a method wherein a candidate substance as thefunctional derivative is mixed with HLA-A24-expressing cells, and thecandidate substance not bound to HLA-A24 molecules is washed away, toreact the resulting product with the CTL. When candidate-specificcytotoxicity, cytokine release or proliferation reaction is recognizedin this method, the candidate substance can be judged to be thefunctional derivative.

The second method is a method wherein a candidate substance is mixedwith cells having an ability to present the antigen, and incubated foran appropriate time, for example, for a time required for the antigen tobe incorporated and processed and for the antigen peptide and HLAmolecule complex to be presented to the surface of the cell, to reactthe resulting product with the CTL. When candidate-specific cytokinerelease or proliferation reaction is recognized in this method, thecandidate substance can be judged to be the functional derivative.

A third method is a method wherein a nucleic acid encoding an amino acidsequence of the candidate substance is bound to an expression vectorcapable of presenting a peptide on HLA-A24 molecules on cells having anability to present the antigen described below, and wherein suitablecells are transformed by the resulting recombinant vector, therebyreacting the resulting cells having an ability to present the antigenwith the CTL. In such method, when candidate substance-specific cytokinerelease or proliferation reaction is recognized, the candidate substancecan be judged to be the functional derivative.

The functional derivative includes, for example, peptides having anability to bind to HLA-A24 molecules to give a complex of the peptideand an HLA-A24 molecule recognized by a CTL recognizing a complex of apeptide represented by SEQ ID NO: 1 or 2 and an HLA-A24 molecule, out ofpeptides having the amino acid sequence of SEQ ID NO: 1 or 2, wherein:

1) a second amino acid residue from the N-terminal is substituted withan amino acid selected from the group consisting of a Tyr residue, a Pheresidue, a Met residue and a Trp residue typical of the peptide bindingto HLA-A24 molecules and/or2) the C-terminal amino acid is substituted with an amino acid selectedfrom the group consisting of a Leu residue, an Ile residue, a Trpresidue and a Phe residue typical of the peptide binding to HLA-A24molecules, in order to enhance binding to HLA-A24 molecules. Morespecifically, the functional derivative is, for example, a peptidewherein the C-terminal amino acid Phe residue is substituted with a Leuresidue in the amino acid sequence of SEQ ID NO: 2.

Those peptides which have an ability to bind to HLA-A24 molecules togive a complex of the peptide and an HLA-A24 molecule recognized by theCTL of the present invention, out of those peptides wherein one orseveral amino acid residues (amino acid residues to be substituted) aresubstituted with amino acid residues or amino acid analogues similar inside chain to the amino acid residues to be substituted in the aminoacid sequence of SEQ ID NO: 1 or 2 are also included.

The “amino acid residues similar in side chain to the amino acidresidues to be substituted” refer to other amino acid residue(s)belonging to the same group as an amino acid residue in any one of thefollowing groups 1 to 7:

1. glycine (Gly) residue and alanine (Ala) residue;2. valine (Val) residue, isoleucine (Ile) residue, leucine (Leu) residueand methionine (Met) residue;3. asparagine (Asn) residue and glutamine (Gln) residue;4. aspartic acid (Asp) residue and glutamic acid (Glu) residue;5. serine (Ser) residue and threonine (Thr) residue;6. lysine (Lys) residue and arginine (Arg) residue; and7. phenylalanine (Phe) residue and tyrosine (Tyr) residue.

A sixth embodiment of the present invention relates to a therapeuticagent for cancer, which comprises the CTL in the fifth embodiment as anactive ingredient. Since the therapeutic agent for cancer of the presentinvention comprises the CTL of the present invention, the therapeuticagent for cancer exhibits an excellent effect that the agent can actspecifically on tumor cells of individuals, especially Asian races,particularly Japanese.

Particularly, the therapeutic agent of the present invention is usefulfor treatment of cancer wherein expression of the antigen recognized bythe CTL in the fifth embodiment, i.e., MAGE-4 or SAGE, is recognized.

The therapeutic agent for cancer according to the present invention isprovided in the form of a suspension of the CTL in a pharmaceuticallyacceptable diluent.

In this specification, the “diluent” refers to, for example, a mediumsuitable for storage of the CTL, or physiologic saline, phosphatebuffered physiologic saline, and the like.

The medium includes, but is not limited to, for example, RPMI, AIM-V,X-VIVO10 and the like.

For the purpose of stabilization, a pharmaceutically acceptable carriermay also be added to the therapeutic agent for cancer according to thepresent invention. Here, the carrier includes, for example, human serumalbumin and the like.

The content of the CTL in the therapeutic agent for cancer according tothe present invention is 1×10⁴ cells/milliliter or more, preferably5×10⁵ cells/milliliter or more, and 1×10⁸ cells/milliliter or less,preferably 5×10⁷ cells/milliliter or less, per kind of CTL.

When the therapeutic agent for cancer according to the present inventionis to be administered to human, the agent can be administered, forexample, with a syringe. The dose of the therapeutic agent for canceraccording to the present invention can be determined suitably dependingon body weight, disease state, and the like of the individual, and thenumber of CTLs administered into an adult is desirably set to be 1×10⁶to 1×10¹⁰ cells per kind of CTL. Here, the above range is a standard andnot restrictive. The active ingredient CTL is a CTL derived from a humanas the subject of administration or a CTL having the same type of HLA asin the human, and thus the toxicity of the therapeutic agent for canceraccording to the present invention is not particularly recognized.

Pharmacological evaluation of the therapeutic agent for cancer accordingto the present invention can be carried out in the same manner asdescribed above.

A seventh embodiment of the present invention relates to an inducer ofthe cytotoxic T lymphocytes (CTLs) in the fifth embodiment, whichcomprises a human major histo-compatibility antigen (HLA)-A24-restrictedantigen peptide represented by SEQ ID NO: 1 or 2, or its functionalderivative, as an active ingredient. The present invention is based onthe present inventors' finding that the peptide consisting of an aminoacid sequence represented by SEQ ID NO: 1 or 2, identified by thepresent inventors as the HLA-A24-restricted antigen peptide recognizedby an HLA-A24-restricted CTL induced against tumor antigen MAGE-A4 orSAGE, is useful for inducing a CTL from human peripheral bloodlymphocytes.

Since the CTL inducer of the present invention comprises theHLA-A24-restricted antigen peptide or the functional derivative thereofas an active ingredient, the CTL inducer of the present inventionexhibits an excellent effect that the CTL in the fifth embodiment can beinduced by a simple technique. Further, the CTL inducer of the presentinvention exhibits an excellent effect that an action can be exertedspecifically on tumor cells in individuals, especially Asian races,particularly Japanese.

The CTL inducer of the present invention is provided in the form of asuspension of the HLA-A24-restricted antigen peptide or the functionalderivative thereof alone or in a mixture thereof with other molecules(helper T cell antigen peptide and/or adjuvant) in physiologic saline orphosphate buffered physiologic saline, or in such a form that thepeptide or the functional derivative thereof alone, or in the mixture,can be suspended at use.

The HLA-A24-restricted antigen peptide or the functional derivativethereof used in the CTL inducer of the present invention may be boundcovalently to a higher fatty acid or a helper T cell antigen peptide ormay be formed into a complex with an HLA-A24 molecule. Desirably, thecontent of the HLA-A24-restricted antigen peptide or the functionalderivative thereof in the CTL inducer of the present invention is 0.01%by weight or more, preferably 0.1% by weight or more and 100% by weightor less, preferably 95% by weight or less, per kind ofHLA-A24-restricted antigen peptide or the functional derivative thereof.

The CTL inducer of the present invention can be utilized as an additivefor a medium for in vitro proliferation of the CTL of the presentinvention; in diagnosis of an immune-sensitized state with T lymphocyteproliferation activity, delayed skin reaction or the like as anindicator; and the like.

When the CTL inducer of the present invention is used, for example, asan additive in a medium, it is desirable that the amount of the CTLinducer of the invention used is, in terms of peptide concentration inthe medium, 1 ng/milliliter or more, preferably 100 ng/milliliter ormore and 100 μg/milliliter or less, preferably 1 μg/milliliter or less,per type of the HLA-A24-restricted antigen peptide or the functionalderivative thereof. The medium includes mediums such as serum-containingRPMI or AIM-V.

The effect of the CTL inducer of the present invention on induction of aCTL can be evaluated, for example, by measuring the activity of the CTLinduced with the inducer, in terms of cytotoxicity on the target cells,proliferation reaction in the presence of antigen-presenting cells, theamount of antigen-specific cytokines released, or the like.

An eighth embodiment of the present invention relates to a therapeuticagent for cancer, which comprises an HLA-A24-restricted antigen peptiderepresented by SEQ ID NO: 1 or 2 or the functional derivative thereof asan active ingredient. Since the therapeutic agent for cancer accordingto the present invention comprises the HLA-A24-restricted antigenpeptide or the functional derivative thereof as an active ingredient,the therapeutic agent for cancer according to the present inventionexhibits an excellent effect that the agent can act specifically ontumor cells in individuals, especially Asian races, particularlyJapanese.

The therapeutic agent for cancer according to the present invention isprovided in the form of:

1) the antigen peptide alone,2) a mixture of the antigen peptide and a pharmaceutically acceptablecarrier and/or diluent, or3) the above-mentioned 1) or 2) to which a subsidiary agent was added ifnecessary.

Here, the carrier includes, for example, human serum albumin and thelike.

The diluent includes, for example, a phosphate buffer, distilled water,physiologic saline, and the like.

Furthermore, the subsidiary agent includes pharmaceutically acceptableadjuvants and the like. The adjuvants include, but are not limited to,for example, (a) Freund complete adjuvant, (b) Freund incompleteadjuvant, (c) inorganic gel such as aluminum hydroxide, alum, (d)surfactants such as lysolecithin, dimethyl octadecyl ammonium bromide,(e) polyanions such as dextran sulfate, poly IC, (f) peptides such asmuramyl peptide, tuftsin, and (g) monophosphoryl lipid (MPL) Amanufactured by Ribi Corporation or functional equivalents thereof.

When the therapeutic agent for cancer according to the present inventionis administered into humans, the therapeutic agent for cancer accordingto the present invention may be administered, for example,subcutaneously, intracutaneously or intravenously with a syringe, or maybe administered by transdermal absorption through a mucosa by a methodsuch as spraying.

The content of the HLA-A24-restricted antigen peptide or the functionalderivative thereof in the therapeutic agent for cancer according to thepresent invention is desirably 0.01% by weight or more, preferably 0.1%by weight or more, and 100% by weight or less, preferably 95% by weight,per type of HLA-A24-restricted antigen peptide or the functionalderivative thereof.

Desirably, the dose of the therapeutic agent for cancer according to thepresent invention per adult is, in terms of peptide concentration, 0.1μg/kg or more, preferably 1 μg/kg or more, and 10 mg/kg or less,preferably 1 mg/kg or less, more preferably 100 μg/kg or less, per typeof HLA-A24-restricted antigen peptide or the functional derivativethereof. Here, upon administration into humans, toxicity of thetherapeutic agent for cancer according to the present invention is notparticularly recognized.

A ninth embodiment of the present invention relates to a tetramer fordetecting a T cell receptor possessed by the CTL in the fifthembodiment, which comprises an HLA-A24-restricted antigen peptiderepresented by SEQ ID NO: 1 or 2 or a functional derivative thereof.Such tetramer binds to TCR possessed by the CTL. The tetramer of thepresent invention comprises the HLA-A24-restricted antigen peptide orthe functional derivative thereof, and thus exhibits an excellent effectthat the CTL can be monitored in individuals, especially Asian races,particularly Japanese. The present invention is based on the presentinventor's finding that a MAGE-A4- or SAGE-specific HLA-A24-restrictedCTL can be detected by a tetramer formed by, with biotin-streptavidin,tetramerizing MHC/antigen peptide complexes prepared from the antigenpeptide of SEQ ID NO: 1 or 2.

A CTL is activated to cause various immune reactions, upon recognitionof, along with the MHC molecule, the antigen peptide binding to the MHCmolecule on the cell surface of an antigen-presenting cell or a targetcell by a complex of a T cell receptor (TCR) and a CD3 molecule on thecell surface of the CTL.

According to the tetramer of the present invention, the MHC tetramer canbe utilized for a different TCR, i.e., an HLA-A24-restricted antigenpeptide from MAGE-A4 or SAGE, and is useful in analysis on behavior orfunctions of a CTL, particularly for a method of specific measurement ofa CTL having the TCR of interest.

The tetramer of the present invention can be obtained, for example, bytetramerizing complexes formed from the HLA-A24-restricted antigenpeptide of SEQ ID NO: 1 or 2 or the functional derivative thereof, β2microglobulin and an HLA-A24 molecule by a biotin-streptavidin method.

The tetramer of the present invention binds to a T cell receptorpossessed by a CTL, and is thus useful, for example, in that thetetramer can be utilized in a method for measuring a CTL which can becarried out in a short time and is simple, by using the formation ofcomplex of the tetramer and a CTL as an indicator in place ofmeasurement of the cytotoxicity of a CTL. The tetramer of the presentinvention is useful particularly in detection or separation of a CTL ina sample such as PBMC containing only a small amount of CTL.

The tetramer of the present invention can also be used in ELISPOT(enzyme-linked immunospot) used for monitoring T lymphocytes in the bodyof a patient, or for target cells in a cytotoxicity test.

The tetramer of the present invention is also useful in monitoring aCTL.

Hereinafter, the present invention will be described in more detail byreference to the Examples, but the present invention is not limited tothe Examples.

Example 1 Identification of HLA-A2402-Restricted CTL Epitope (AntigenPeptide) using HLA-A2402 Transgenic Mice (1) Materials and Method

HHDA2402+/−β2m−/− mice

A DNA construct (HHDA2402) containing an HLA-A2402 leader sequence,human β2 microglobulin, HLA-A2402 α1 and α2 domains, an H-2D^(b) α3transmembrane domain, and a cytoplasm domain was constructed. Theresulting construct was cloned into an expression vector pcDNA 3.1(manufactured by Invitrogen Corporation). The HHDA2402 construct (4 kbSalI-NotI fragment) was injected into fertilized eggs of C57BL/6 mice,to give HHDA2402-expressing mice. Then, the HHDA2404-expressing micewere bred with β2m−/− mice (manufactured by The Jackson Laboratory). Theresulting HHDA2402+/−β2 m+/− were bred with β2m−/− mice to giveHHDA2402+/−β2m−/− mice (referred to hereinafter as “HHDA2402 mice”).

(2) Cell Strain

TAP transporter-deficient strain T2 (J. Immunol., 167, p. 2529-2537(2001)) was transfected with an HLA-A2402 cDNA to prepare T2A24 strain.The following cell strains were also used in preparation of CTL targetcells: breast cancer cell strain R27 (A2402-negative), esophagus cancerstrain KE-4 (A2402-positive), esophagus cancer strain TE-10(A2402-positive), chronic myelocytic leukemia strain K562(A2402-negative), lung cancer strain 11-18 (A2402-positive) andembryonic renal cell 293 (A2402-negative). The above-mentioned R27, K562and 293 were transfected with an HLA-A2402 cDNA to prepare R27A24,K562A24 and 293A24. Human B-lymphoblast (LCL) was prepared in a usualmanner from HLA-A2402-positive or negative cells derived from volunteersby using EBV.

(3) Plasmid

cDNA of the full-length MAGE-A4, SAGE or EBNA-3A was cloned into pcDNA3.1.

(4) Immunization with a Gene Gun

Using Helios Gene Gun System (trade name, manufactured by Bio-RadLaboratories, Inc.), gold particles coated with plasmid DNA wereadministered endoabdominally at a helium pressure of from 350 to 400 psiinto HHDA2402 mice (6-to 8-week-old, female). The gold particles wereprepared according to a manufacturer's manual. After 2 weeks, boosteradministration was carried out. After 1 week, spleen cells werecollected.

One week after the final immunization, MACS system (trade name,manufactured by Mitlenyi Biotec) using CD8 alpha (Lyt 2) microbeads wasused, to select CD8-positive T cells. The purity of the resulting T cellfraction analyzed by flow cytometry was 95% or more.

(6) ELISPOT Assay (Mice)

A 96-well nitrocellulose ELISPOT plate (trade name: MAHA 54510,manufactured by Millipore Corporation) was incubated overnight with 2μg/milliliter anti-mouse IFN-gamma mAb (trade name: clone R4-6A2,manufactured by PharMingen Company) at 4° C., to coat the plate. Eachwell was washed with phosphate buffered physiologic saline (PBS). Thewell after washing was blocked by incubation at 37° C. for 2 hours inFCS-containing RPMI1640 medium.

Fresh CD8-positive cells (1×10⁵ cells/well) derived from the immunizedmice and CD8-negative cells (1×10⁶ cells/well) pulsed with variouspeptides were seeded to each well and regulated so as to have a finalvolume of 200 μl. Then, the cells were cultured at 37° C. for 22 hours.Thereafter, the cells were washed sufficiently with PBS containing 0.05wt % Tween™ 20 (referred to as “PBS-Tween”). Two hundreds microliters of1.25 μg/milliliter biotinylated anti-mouse IFN-gamma mAb (trade name,manufactured by PharMingen Company) was added to the cells afterwashing, and the cells were then cultured overnight at 4° C. The cellsthus obtained were washed with PBS-Tween. One hundred microliters of 1μg/milliliter streptavidin-alkaline phosphatase conjugate (trade name,manufactured by Mabtech Ltd.) was added to the cells after washing.Thereafter, the resulting mixture was incubated at room temperature for90 minutes, to carry out reaction. The resulting product was washed withPBS-Tween and then stained with an alkaline phosphatase conjugatesubstrate kit (trade name, manufactured by Bio-Rad Laboratories, Inc.).Thereafter, the product after staining was washed with distilled waterto terminate the reaction. Then, the plate was dried and spots werecounted.

(7) Estimation of Epitope Peptides

Nine-residue peptide which might have an ability to bind to HLA-A2402were searched for by using HLA Peptide Binding Predictions running on aweb site of BioInformatics & Molecular Analysis Section (BIMAS).

Each of five peptides derived from SAGE and ten peptides derived fromMAGE-A4 shown in Table 1 below were chemically synthesized by a peptidesynthesizer.

TABLE 1 Antigen Amino acid name position Amino acid sequence SAGE841-848 NYERIFILL (SEQ ID NO: 3) 776-784 LYKPDSNEF (SEQ ID NO: 4)715-723 LYATVIHDI (SEQ ID NO: 2) 621-629 QYAAVTHNI (SEQ ID NO: 5)250-258 TYNVPEEKM (SEQ ID NO: 6) MAGE-A4 239-247VYGEPRKLL (SEQ ID NO: 7) 143-151 NYKRCFPVI (SEQ ID NO: 1) 271-279EFLWGPRAL (SEQ ID NO: 8) 151-159 IFGKASESL (SEQ ID NO: 9) 113-121KVDELAHFL (SEQ ID NO: 10) 283-291 SYVKVLEHV (SEQ ID NO: 11) 124-132KYRAKELVT (SEQ ID NO: 12) 199-207 KTGLLIIVL (SEQ ID NO: 13) 301-309AYPSLREAA (SEQ ID NO: 14) 43-51 SSPLVPGTL (SEQ ID NO: 15)

Here, the “amino acid position” in the table indicates the position ofeach peptide in an amino acid sequence of SAGE or MAGE-A4.

Furthermore, similarly, EB virus-derived EBNA 3A₂₄₆₋₂₅₄ (SEQ ID NO: 16,RYSIFFDYM) and HER2-derived HER2₆₃₋₇₁ (SEQ ID NO: 17, TYLPTNASL) weresynthesized. The purity of the peptides used was 90% or more.

HHDA2402 mice were immunized with an expression plasmid carrying EBvirus-derived EBNA 3A gene by a gene gun. After immunized twice,spleen-derived CD8+ T cells were prepared. CD8-negative cells pulsedwith the EBNA 3A₂₄₆₋₂₅₄ peptide were used as target cells, to carry outELISPOT assay. The results are shown in FIG. 1A.

As a result, EBNA3A₂₄₆₋₂₅₄ peptide-specific CTLs were detected as shownin FIG. 1A.

A tumor antigen MAGE-A4-derived candidate peptide and a testis antigenSAGE-derived candidate peptide (Table 1 above) were then subjected toELISPOT assay in the same manner. The results are shown in FIGS. 1B and1C.

As a result, two peptides (MAGE-A4₂₃₉₋₂₄₇ and MAGE-A4₁₄₃₋₁₅₁) werepositive in MAGE-A4, as shown in FIG. 1B. Similarly, as shown in FIG.1C, two peptides (SAGE₇₇₆₋₇₈₄ and SAGE₇₁₅₋₇₂₃) were positive in SAGE.

Wild-type C57BL6 mice were then used, to conduct the same experiment asabove. As a result, only MAGE-A4₂₃₉₋₂₄₇ and SAGE₇₇₆₋₇₈₄ were positive.

Accordingly, MAGE-A4₁₄₃₋₁₅₁ and SAGE₇₁₅₋₇₂₃ were identified as antigenpeptides presented by HLA-A2402.

Example 2 Preparation of HLA-2402 Antigen Peptide-Specific HumanCytotoxic T Lymphocytes (CTLs) Using CD4-Positive PHA Blast Cells intowhich mRNA was Introduced

(1) Preparation of mRNA

MAGE-A4 plasmid and SAGE plasmid were linearized. The resulting productsand T7 polymerase (trade name: mMESSAGE mMACHINE T7 Kit, manufactured byAmbion, Inc.) were used, to conduct in vitro transfer according to amanufacturer's manual. Thereafter, the resulting product waspolyadenylated with poly A polymerase (trade name: Poly(A) Tailing Kit,manufactured by Ambion, Inc.) according to a manufacture's manual. Theresulting RNA was stored at −80° C. until use.

(2) Preparation of CD4-Positive Phytohemagglutinin (PHA) Blast Cells

By using positive selection using MACS CD4 microbeads (manufactured byMiltenyi Biotec), fresh CD4-positive cells were separated from PBMC. Theresulting CD4-positive cells were seeded on a 24-well plate(manufactured by Corning Incorporated) at a cell density of from 1 to2×10⁶ cells/milliliter RPMI-1640 medium (composition: containing 25 mMHepes, 10 wt % inactivated human AB serum, 2 mM L-glutamine, 100U/milliliter penicillin, 100 μg/milliliter streptomycin) per well.

On the 0th day, PHA (trade name: HA15, manufactured by Murex S.A.) wasadded to the medium in each well of the plate at a final concentrationof 10 μg/milliliter. On the 3rd day, half amount of the medium wasexchanged with the above medium containing IL-2 (20 U/milliliter) andIL-7 (40 ng/milliliter). Exchange of the medium was repeated every 3days, whereby activated CD4-positive cells were obtained. The mRNA wasintroduced by electroporation into the activated CD4-positive cells at14 to 28 days after culture was initiated. The resulting cells were usedbelow as antigen-presenting cells.

(3) In Vitro Induction of Human CTLs Using mRNA-Introduced CD4-PositiveBlast Cells

MACS CD8 Microbeads (trade name, manufactured by Miltenyi Biotec) wereused, to separate CD8+ T cells from PBMC. The number 5×10⁵ cells of CD8+T cells thus obtained were stimulated with radiation (30 Gy)-irradiated1×10⁵ mRNA-introduced CD4+ PHA blast cells in a 96-well round plate(manufactured by Nunc Corporation). After 7 days, the CD8+ T cells werestimulated again with radiation (30 Gy)-irradiated 1×10⁵ mRNA-introducedCD4+ PHA blast cells, and then cultured in an IL-2 (20IU/milliliter)-containing RPMI1640 medium for 7 days.

(4) In Vitro Amplification of CTLs

To amplify the sensitized CD8+ T cell group containing theantigen-specific CTLs, autologous LCLs (5×10⁶ cells) into which thetarget antigen mRNA was introduced, autologous PBMCs (2.5×10⁷ cells),and IL-2 (20 IU/milliliter) were added to the sensitized CD8+ T cellgroup. The resulting mixture was cultured in the absence of anti-CD3antibody in a 25-milliliter flask (manufactured by CorningIncorporated).

(5) Limiting Dilution

The CD8+ T cells were diluted to a density of 0.3 cell/well in a 96-wellround plate (manufactured by Nunc Corporation). Furthermore, autologousPBMCs (5×10⁴ cells/well), radiation-irradiated LCLs (1×10⁴ cells/well),IL-2 (20 IU/milliliter), and anti-CD3 mAb (30 ng/milliliter) were addedto the resulting dilution. The resulting mixture was cultured. As aresult, CD8+ T cells lyzing the antigen-presenting cells as the targetwere proliferated in the presence of radiation-irradiated PBMC,radiation-irradiated LCL, and anti-CD3 mAb.

(6) ELISPOT Assay (Human)

A 96-well nitrocellulose ELISPOT plate (trade name: MAHA 54510,manufactured by Millipore Corporation) was coated by overnightincubation at 4° C. with 2 μg/milliliter antihuman IFN-γ mAb (tradename: 1-D1K). Each well was washed with PBS, and then blocked byincubation at 37° C. for 2 hours with RPMI1640 medium containing 10 wt %human AB serum. Effector cells (2×10⁴ cells/well) and peptide-pulsedT2A24 cells (5×10⁴ cells/well) were seeded to each well. The cells inthe well were cultured at 37° C. for 18 hours. The resulting cells werewashed sufficiently with PBS-Tween. After washing, the cells wereincubated overnight with 1.25 μg/milliliter biotinylated anti-mouseIFN-gamma mAb (manufactured by PharMingen Company) at 4° C. Theresulting product was washed with PBS-Tween. One hundred microliters of1 μg/milliliter streptavidin-alkaline phosphatase conjugate(manufactured by Mabtech Ltd.) was added to the resulting product andreacted by incubation at room temperature for 60 minutes. The resultingproduct was washed with PBS-Tween and then stained with an alkalinephosphatase conjugate substrate kit (trade name, manufactured by Bio-RadLaboratories, Inc.). The resulting product was then washed withdistilled water to terminate the reaction. Then, the plate was dried andspots were counted.

(7) ⁵¹Cr Release Cytotoxicity Assay

The cytotoxicity was evaluated in a usual manner as follows. The targetcells were labeled with 100 μCi (3.7×10⁶ Bq) ⁵¹Cr. Then, 1×10⁴ targetcells were reacted in a 96-well V-bottomed plate (manufactured by NuncCorporation) with effector cells at various density at 37° C. After 5hours, 100 μl supernatant was collected and measured for radioactivity.This measurement was conducted in triplicate, and the average ofspecific lysis % in 3 wells was calculated on the basis of the followingformula:

Specific cytotoxic activity (%)=[(measurement of each well−minimumrelease level)/(maximum release level−minimum release level)]×100

In the above formula, the “minimum release level” is the ⁵¹Cr level ofthe well containing only the target cells and K562 cells, and indicatesthe natural release level of ⁵¹Cr from the target cells. The “maximumrelease level” is indicative of the ⁵¹Cr release level upon disruptionof the target cells with surfactant Triton™ X-100 added to the cells.

According to the above-mentioned items (1) to (7), the followingevaluation was conducted.

CD8+ cells prepared from healthy normal humans were sensitized in vitrowith autologous CD4+ PHA blast cells into which mRNA was introduced. TheCD8+ cells were stimulated twice with the autologous CD4+ PHA blastcells into which MAGE-A4 mRNA was introduced. The results of ELISPOTassay are shown in FIG. 2.

As a result, MAGE-A4-specific bulk CTLs were obtained as shown in panelA in FIG. 2. As shown in panel B in FIG. 2, cells obtained by amplifyingthe MAGE-A4-specific bulk CTLs with mRNA-introduced autologous LCL,autologous PBMC and IL-2 showed reaction specific to T2A24 cells pulsedwith MAGE-A4₁₄₃₋₁₅₁ peptide.

Furthermore, MAGE-A4₁₄₃₋₁₅₁-specific #2-28 cells obtained by limitingdilution were analyzed by flow cytometry using MAGE-A4₁₄₃₋₁₅₁ tetramerand anti-CD8 antibody, and the results are shown in panels A and B inFIG. 3. The cytotoxicity by MAGE-A4₁₄₃₋₁₅₁-specific #2-28 cells wasexamined, and the results are shown in panels C and D in FIG. 3.

As a result, the #2-28 cells obtained by limiting dilution were stainedpositively with the MAGE-A4₁₄₃₋₁₅₁ tetramer, but not stained with atetramer used as a control, as shown in panels A and B in FIG. 3. Asshown in panel C in FIG. 3, the #2-28 cells showed HLA-A2402-restrictedcytotoxicity on the target cells pulsed with the MAGE-A4₁₄₃₋₁₅₁-peptide.Furthermore, as shown in panel D in FIG. 3, the #2-28 cells showedcytotoxicity on the tumor cell strain expressing both MAGE-A4 andHLA-A2402. This indicates that the MAGE-A4₁₄₃₋₁₅₁ peptide is presentednot only against the mRNA-introduced CD4+ PHA blast cells but alsoagainst the HLA-2402-positive tumor cells by intracellular processing ofMAGE-A4 antigen.

Similarly, human bulk CTL cells were obtained by sensitization twicewith SAGE mRNA-introduced autologous CD4+ PHA blast cells. IFN-γ ELISPOTassay was conducted using the resulting human bulk CTL cells. As thetarget cells, T2A24 cells pulsed with SAGE₇₁₅₋₇₂₃ peptide or T2A24 cellspulsed with the control peptide were used. The results are shown in FIG.4A.

As a result, SAGE₇₁₅₋₇₂₃-specific bulk CTLs were inducted by stimulationtwice with CD4+ blast cells into which truncated SAGE mRNA wasintroduced, as shown in FIG. 4A.

The bulk CTLs were amplified in vitro. The resulting CTL was analyzed byflow cytometry using SAGE₇₁₅₋₇₂₃ tetramer and anti-CD8 antibody. Theresults are shown in FIG. 4B.

As a result, the resulting bulk CTL was positive in staining withSAGE₇₁₅₋₇₂₃ A24 tetramer, as shown in FIG. 4B. Accordingly, it wasrevealed that bulk CTLs contained CD8+ cells positive to SAGE₇₁₅₋₇₂₃HLA-A24 tetramer.

T2A24 cells were then pulsed with SAGE₇₁₅₋₇₂₃ peptide. As the control,the cells were pulsed with HER2₆₃₋₇₁ peptide. Thereafter, the pulsedcells were subjected to ELISPOT assay. The results are shown in FIG. 4C.

As a result, IFN-γ was released only when T2A24 pulsed with SAGE₇₁₅₋₇₂₃peptide was used as the target cell, as shown in FIG. 4C.

SAGE₇₁₅₋₇₂₃-specific HLA-A24 CTL cells #22 (#22 cells) were prepared bylimiting dilution. The resulting #22 cells were analyzed by flowcytometry using SAGE₇₁₅₋₇₂₃ HLA-A24 tetramer and anti-CD8 antibody. Theresults are shown in FIG. 5A. The cytotoxicity by the #22 cells was alsoexamined. The results are shown in FIG. 5C.

As a result, the #22 cells were positive to SAGE₇₁₅₋₇₂₃ HLA-A24tetramer, as shown in FIG. 5A. When the #22 cells were co-cultured with293-A2402 into which a plasmid carrying the full length of SAGE gene wasintroduced, the #22 cells secreted IFN-γ. The #22 cells (1×10⁴ cells),and 293A24 cells (1×10⁴ cells) transformed with SAGE cDNA, were culturedin a 96-well round plate for 18 hours. As a result, IFN-γ was releasedas shown in FIG. 5B. As shown in panels A and B in FIG. 5C, the #22cells showed cytotoxicity to both K562A24 and R27A24. Here, both theK562A24 and R27A24 expressed HLA-A2402 and SAGE. Furthermore, as shownin panel C in FIG. 5C, there was specific cytotoxicity on A2402+LCLcells into which mRNA of SAGE gene was introduced.

Example 3 SAGE₇₁₅₋₇₂₃-Specific CD8+ T Cells are Induced fromA2402-Positive Healthy Normal Humans with High Probability

In Vitro Induction of Human CTLs Using CD8− PBMC Pulsed with a Peptide

The number 1×10⁷ of CD8-negative PBMC were pulsed with 10 μM peptide byincubation at room temperature for 1 hour and at 37° C. in 5% CO2 for 1hour, in 200 μl RPMI1640 medium containing 25 mM Hepes, 10 wt %inactivated human AB-positive serum, 2 mM L-glutamine, 100 U/milliliterpenicillin, and 100 μg/milliliter streptomycin. The resulting cells wereused as antigen-presenting cells. The number 5×10⁵ of CD8+ T cells thatwere separated were then stimulated by incubation for from 10 to 12 dayswith 1×10⁶ cells of the peptide-pulsed CD8− PBMC. On Days 1, 4 and 7,half amount of the medium was exchanged with fresh one, and human IL-2(20 IU/milliliter) and IL-7 (50 ng/milliliter) were added thereto.Culturing for induction was conducted in 200 μA RPMI1640 medium(containing 25 mM Hepes, 10 wt % inactivated human AB serum, 2 mML-glutamine, 100 U/milliliter penicillin, 100 μg/milliliterstreptomycin).

It was examined whether an HLA-A2402-restricted SAGE₇₁₅₋₇₂₃-specific CTLwas induced in vitro by once stimulating CD8+ cells fromHLA-A2402-positive healthy normal volunteers, with CD8-negative PBMCpulsed with SAGE₇₁₅₋₇₂₃ peptide.

As a result, SAGE₇₁₅₋₇₂₃ HLA-A24 tetramer-positive T cells were detectedin three out of six HLA-A2402+ healthy normal humans at 10 days aftermixed lymphocyte reaction in vitro. FIGS. 6A to 6D each show one exampleof the analysis results.

Example 4 Preparation of a Tetramer and Flow Cytometry Analysis

HLA-A2402 heavy chain and β2-microglobulin were expressed as aninsoluble polymer in Escherichia coli. Here, in order to add a sequenceserving as a substrate for biotinylating enzyme BirA to the C-terminalof the HLA-A2402 heavy chain, a corresponding polynucleotide was addedto the nucleic acid encoding the HLA-A2402 heavy chain. A monomerHLA/β2-microglobulin/peptide complex was prepared in vitro by foldingthe insoluble polymer in the presence of MAGE-A4₁₄₃₋₁₅₁ peptide orSAGE₇₁₅₋₇₂₃. The resulting product was biotinylated with a recombinantBirA enzyme (manufactured by Avidity) and tetramerized withphycoerythrin-labelled streptavidin (manufactured by Molecular ProbesInc.) to give an MHC/peptide tetramer.

In staining, sensitized CD8+ T cells were reacted with 20 μg/millilitertetramer at 37° C. for 30 minutes, and then reacted with a tricoloranti-CD8 monoclonal antibody (trade name, Caltag Laboratories,Burlingame, Calif., USA) on ice for 15 minutes. After washing, thestained cells were analyzed by flow cytometry (trade name: FACSCalibur,manufactured by Becton Dickinson, and Company). As a result, it wasconfirmed that the prepared tetramer was bound to the sensitized CD8+ Tcells.

EQUIVALENTS

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential characteristics thereof. Thepresent invention is therefore to be considered in all respects asillustrative and not restrictive. The scope of the invention isindicated by the appended claims rather than by the foregoingdescription. Furthermore, all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

1. An RNA-introduced T lymphocyte into which an RNA encoding an antigen of interest is introduced, which has an activity to induce a T lymphocyte which recognizes the antigen.
 2. The RNA-introduced T lymphocyte according to claim 1, which is a CD4-positive cell activated with phytohemagglutinin.
 3. A method for inducing a T lymphocyte, comprising using, as an antigen-presenting cell, an RNA-introduced T lymphocyte into which an RNA encoding an antigen of interest is introduced, to induce a T lymphocyte which recognizes the antigen.
 4. The method according to claim 3, wherein the RNA-introduced T lymphocyte is a CD4-positive cell activated with phytohemagglutinin.
 5. The method according to claim 3, wherein the T lymphocyte which recognizes the antigen is a cytotoxic T lymphocyte positive to CD8.
 6. The method according to claims 3, wherein the antigen is a tumor-associated antigen or an antigen of an infectious microorganism.
 7. The method according to claim 6, wherein the RNA is an RNA prepared from a cancer tissue.
 8. The method according to claim 6, wherein the antigen is EBNA3A and/or CMVpp65.
 9. An immune inducer containing the RNA-introduced T lymphocyte as defined in claim
 1. 10. A therapeutic agent for cancer or infectious disease, containing the T lymphocyte obtained by the method of claim 3 as an active ingredient.
 11. A cytotoxic T lymphocyte which recognizes a cell presenting on the surface thereof a complex of a human major histo-compatibility antigen (HLA)-A24-restricted antigen peptide represented by SEQ ID NO: 1 and an HLA-A24 molecule or a complex of a functional derivative of the HLA-A24-restricted antigen peptide and an HLA-A24 molecule, and which is CD8-positive.
 12. A therapeutic agent for cancer, containing the cytotoxic T lymphocyte of claim
 11. 